The intracellular molecular mechanisms underlying the regulation of the AMPA receptor have been dramatically elucidated in the past few decades. In contrast, the regulation of the extracellular domain remains unclear. Here, we focused on N-glycosylation of the AMPA receptor in the extracellular domain and tried to clarify their functions by combining molecular biological and electrophysiological techniques.
First, we examined the effects of PNGase F, which is a glycosidase that cleaves the N-glycosylation site at an asparagine residue, on AMPA currents in primary hippocampal cultured neurons and HEK293 cells expressing GluA1 using a whole-cell patch-clamp technique. Glutamate response elicited with puff application demonstrated that the digestion of N-glycosylation induced the re-sensitization of AMPA currents in the both. It is reported that six putative N-glycosylation sites are located in the extracellular domain of GluA1. Next, we performed mutation studies of each N-glycosylation site of GluA1 to identify the N-glycosylation site responsible for the re-sensitization induced by PNGase F treatment. These results indicated that N401 was a critical site for the expression of re-sensitization. Finally, we analyzed the possibility of re-sensitization after PNGase F treatment using acute brain slices. Single electrical stimulation of Schaffer collateral did not show the re-sensitization in hippocampal pyramidal neurons, however, paired pulse stimulation generated the re-sensitization.
Taken together, these results suggest that N-glycosylation, especially at N401 site, can modulate AMPA receptor channel properties.